The biosynthesis of thymine (a DNA base) is essential in all organisms. The last step in this biosynthesis in humans and other eukaryotes is catalyzed by thyA/TYMS-encoded thymidylate synthase (TSase), and its cofactor is recycled by the folA-encoded dihydrofolate reductase (DHFR). In several human pathogens, e.g., those causing anthrax, tuberculosis, typhus, and more, the thyX-encoded flavin-dependent thymidylate synthase (FDTS) provides an alternative biosynthetic path to thymine. At first glance, FDTS seems merely to combine the activities of TSase and DHFR; it has same reactants and products as bi-functional TSase-DHFR. However, FDTS has very different genetic, structural, and mechanistic properties than its human counterparts. The catalytic mechanism of FDTSs is not understood; they have no known potent inhibitors; and inhibitors of classical TSases or DHFRs do not efficiently inhibit FDTSs. Were their mechanism known, rational inhibitor design could lead to new classes of antibiotic drugs with the potential for low toxicity. This proposal aims at studies of the chemical mechanism of FDTS catalysis. This study is of broader interest as preliminary studies suggested that FDTS chemical mechanism is different from that of either bifunctional TSase- DHFRs or any other known mechanism of nucleotide methylation. The proposed studies will employ a broad arsenal of methodologies, including isotopic labeling, single-turnover trapping of reaction intermediates, pre- steady-state and steady-state enzyme kinetics, time-resolved ESI-MS, mutagenesis, alternative cofactors, X-ray crystallography, and the synthesis and testing of putative intermediates. The findings from these diverse mechanistic studies present will test various proposed mechanisms and will illuminate the enigmatic mechanism of this enzyme. Four specific aims are proposed: Specific Aim 1: Trapping and Identification of Intermediates. Specific Aim 2: Examination of the putative exocyclic methylene intermediate. Specific Aim 3: Structural studies. Specific Aim 4: Using 5-deaza-FADH2 as a mechanistic tool.